PROJECT SUMMARY/ABSTRACT Alcohol use disorders (AUDs) are characterized by compulsive and uncontrolled alcohol use, leading to social and occupational impairments. While genetic variation can result in an increased risk of AUDs, chronic alcohol consumption can independently lead to alcohol tolerance and dependence. However, it is unknown how chronic alcohol consumption alters gene expression and leads to neuroadaptations underlying AUDs. There is evidence that small noncoding microRNAs (miRNAs), which regulate target gene (or mRNA) expression at the posttranscriptional level, are abundant in the brain and play important roles in a variety of biological processes such as neuronal differentiation and synapse formation and plasticity. Additionally, each miRNA can regulate the expression of a number of different target mRNAs and each mRNA can be targeted by different miRNAs. Thus, brain miRNAs that regulate the expression of alcohol-responsive mRNAs may act upstream of alcohol- induced neuroadaptations. The objective of the proposed study is to identify dysregulated miRNAs and their target mRNAs in the brains of AUD subjects and generate AUD-associated miRNA-mRNA regulatory networks. The central hypothesis is that AUD-associated miRNAs interact with target mRNAs in reward-related brain regions, forming miRNA-mRNA regulatory networks that are critical for AUD development. This hypothesis will be tested by pursuing three specific aims: (1) identify differentially expressed miRNAs and mRNAs in at least eight reward-related brain regions (prefrontal cortex, nucleus accumbens, ventral tegmental area, hippocampus, amygdala, putamen, caudate, and cerebellum) of AUD subjects using next-generation sequencing (miRNA-Seq and mRNA-Seq); (2) generate AUD-associated miRNA-mRNA regulatory networks in reward-related brain regions using integrated bioinformatics analyses; and (3) refine AUD-associated miRNA- mRNA regulatory networks by validating the predicted miRNA-mRNA pairs using experimental approaches, such as high throughput 3' UTR reporter assays in cell lines and miRNA-mRNA interaction analyses in human embryonic stem cell (hESC)-derived GABAergic cortical neurons (as an in vitro cellular model). The proposed research is significant because it will identify and validate AUD-associated miRNA-mRNA regulatory networks in reward-related brain regions, thus improving our understanding of the epigenetic mechanisms of AUDs. The proposed research is innovative because advanced technologies (such as next-generation sequencing, state- of-the-art bioinformatics programs for miRNA-mRNA network construction, high-throughput 3' UTR reporter assay, and miRNA-mRNA interaction modeling in stem cell-derived neurons) will be used. Our long-term goal is to develop novel pharmacological treatment for AUDs by targeting specific miRNAs and their target genes.